Novel screening methods for inhibitors of the human ubiquitin-conjugating enzymes

dc.contributor.advisor

Auer, Manfred

dc.contributor.advisor

Walkinshaw, Malcolm

dc.contributor.author

Koszela, Joanna

dc.date.accessioned

2016-11-16T11:44:54Z

dc.date.available

2016-11-16T11:44:54Z

dc.date.issued

2014-06-28

dc.identifier.uri

http://hdl.handle.net/1842/17893

dc.description.abstract

The ubiquitin-proteasome system (UPS) controls the stability, activity and localisation of
most of the proteome and regulates virtually all cellular processes through modification of
proteins with ubiquitin. Ubiquitin conjugation is mediated by a conserved enzymatic cascade
composed of E1, E2 and E3 enzymes, which cooperate to activate and transfer ubiquitin to
substrate proteins. Dysfunction of the UPS is implicated in many disease states, including
cancer, neurodegeneration, immune and cardiovascular disorders. Despite the central role of
the UPS in cellular regulation, our understanding of the function, interactions and specificity
of proteins that comprise the UPS is still limited. One approach to dissect and to study the
UPS is to identify molecular probes, which can be used to specifically interrogate catalytic
mechanisms and can be potentially considered as entry points for drug discovery. This work
focuses on developing novel high-throughput screening methods for inhibitors of the
ubiquitin-conjugating enzymes (E2s), using a unicellular organism Saccharomyces
cerevisiae and in vitro technologies.
S. cerevisiae is a model organism, commonly used in research as a valuable tool for genetic
investigations and other high-throughput studies. In this work, we evaluated the toxicity of
exogenously expressed human E2s on yeast cells and discovered that one of the E2s, Ube2U,
significantly inhibited yeast growth. This inhibition was dependent on the Ube2U ubiquitin-conjugation
activity, as demonstrated with a catalytically inactive Ube2U C89A control,
which did not affect yeast growth. The growth defect induced by Ube2U allowed us to
develop a screening setup for inhibitors of Ube2U, where the enzyme activity was coupled to
cell growth readout. Potential Ube2U inhibitors would be identified as rescuers of the slow
growing Ube2U-expressing yeast phenotype.
Although screening methods in yeast are relatively straightforward to set up and run, the
advantages of this system, namely simplicity of the detection signal and high-throughput, are
limited by the fact that yeast is not a recognised large scale screening system in
pharmaceutical industry, and that it is difficult to identify the target in a complex pathway
such as the UPS. In vitro technologies are needed to provide the necessary structure-activity
relationship for chemical optimisation. Therefore, we developed a novel, fluorescence-based,
miniaturised assay technology, suitable for biochemical investigations and screening for
inhibitors of a wide range of specific ubiquitination reactions within the UPS.

en

dc.contributor.sponsor

Wellcome Trust

en

dc.language.iso

en

en

dc.publisher

The University of Edinburgh

en

dc.subject

ubiquitination

en

dc.subject

E2 enzymes

en

dc.subject

high-throughput screening

en

dc.title

Novel screening methods for inhibitors of the human ubiquitin-conjugating enzymes